1. Field of the Invention
The present invention relates to a composition for reprogramming somatic cells to generate induced pluripotent stem cells, comprising Oct4 in combination with Bmi1 or its upstream regulator and a method for generating induced pluripotent stem cells using the same. More particularly, the present invention relates to a technique in which when Oct4 is introduced, in combination with a reprogramming factor selected from among Shh (Sonic hedgehog), oxysterol and purmorphamine, they act in cooperation to induce the somatic cells to undergo a reprogramming process to generate induced pluripotent stem cells.
2. Description of the Related Art
Stem cells are characterized by self-renewal, that is, the ability to go through numerous cycles of cell division while maintaining a state of undifferentiation, and potency, that is, the capacity to differentiate into specialized cell types under suitable conditions. Potency specifies the differentiation potential of the stem cell, and is generally divided into pluripotency, multipotency and unipotency. Therefore, the technique of allowing the stem cells to undergo self-renewal in cell cultures and transforming them into specialized cells has high potential in the cell therapy of various diseases.
Present in adults, various stem cells including hematopoietic stem cells, bone marrow stem cells, and neural stem cells can be used in medical therapies without inducing immune rejection responses because they can be isolated from the patients themselves. In addition, cell therapy with adult stem cells solves the difficulty of securing donors for organ implantation.
Thus far, adult stem cells have been known to remain multipotent, that is, able to differentiate into a number of cells, but only those of a closely related family of cells. Many reports are to the effect that stem cells isolated from the central nervous system (Science 255, 1707-1710 1992; Science 287, 1433-1438 2000), the bone marrow (Science 276, 71-74, 1997; Science 287, 1442-1446, 2000; Science 284, 143-147, 1999), the retina (Science 287, 2032-2036, 2000) and the skeletal muscles (Proc. Natl. Acad. Sci. USA 96, 14482-14486, 1999; Nature 401, 390-394, 1999) are transformed into closely related tissue cells. For example, hematopoietic stem cells can be differentiated into blood-related cells, neural stem cells into neurons or glial cells, and bone marrow stem cells into mesodermal cells. Further, although theoretically they undergo infinite self-renewal adult stem cells are in practice difficult to proliferate in vitro. Moreover, practical limitations are imparted to the isolation of a number of cells from patients.
Pluripotent stem cells are a wonderful resource overcoming the drawbacks of adult stem cells. Pluripotent stem cells can differentiate into nearly any cell and are allowed to replicate infinitely in vitro. Among the pluripotent stem cells known thus far are embryonic stem cells, embryonic germ cells and embryonic carcinoma cells, with most studies focusing on embryonic stem cells for differentiation into specific cells, functionality in animal models of diseases, and therapeutic potency for various diseases.
Nonetheless, the clinical use of embryonic stem cells, like adult stem cells, encounters barriers that must be overcome. Above all, because isolating embryonic stem cells results in the death of the fertilized human embryo, this raises ethical issues. Also, there is the problem of immunological rejection when differentiated cells derived from embryonic stem cells are implanted into patients.
Various attempts have been made to overcome the above-mentioned problems. The greatest amount of attention has been paid to reprogramming differentiated cells into pre-differentiated cells, inter alia. Reprogramming is a generic term expressing the induction of differentiated cells to dedifferentiate into pluripotent stem cells such as embryonic stem cells, generally achieved by nuclear transfer, cell fusion, cell extract treatment, and induced pluripotent stem (iPS) cell technology (Cell 132, 567-582, 2008).
The iPS cell technology succeeded in generating cells closer to embryonic stem cells than has any other technology. Since 2006 in which iPS cells were first produced, a significant number of research articles have been issued. In principle, stem cells similar to embryonic stem cells, e.g., iPS cells, are derived by transfection of four genes (reprogramming inducing genes: Oct4, Sox2, Klf4, and C-Myc/Oct4, Sox2, Nanog, and Lin28) into mouse or human somatic cells, followed by culturing for a long period of time under conditions specialized for embryonic stem cells. These iPS cells have been shown to resemble embryonic stem (ES) cells in their gene expression profile, epigenetic status, in-vitro and in-vivo differentiation into all three germ layers, teratoma formation, chimeric mouse generation, and chimera's competency for germline transmission (Cell 126, 663-676, 2006; Science 318, 1917-1920, 2007).
However, the understanding of the molecular mechanisms underlying reprogramming is meager, which is largely attributed to the use of too many gene factors. To realize the full potential of iPS cells in practical clinical use, it will be essential to improve the reprogramming technology, although established, and to evaluate each generated iPS cell line for safety and efficacy.
Recent research reports have it that the inactivation of the tumor suppressor gene p53 markedly increases the efficiency of iPS cell generation (Nature 460, 1132-1135, 2009). p19Arf and p16Ink4a, both encoded by alternative reading frames of Arf/Ink4a locus, are known to induce the expression of p53 and Rb, respectively. By reducing the expression of both p16Ink4a and p19Arf, iPS cell formation was increased relative to that attained by reducing the expression of p19Arf alone (Nature, 460, 1140-1144, 2009).
Polycomb group (PcG) proteins are epigenetic gene silencers. Bmi1, one of the PcG proteins, is involved in the down-regulation of both p16Ink4a and p19Arf, which leads to suppressing the expression of p53 and Rb (Genes Dev, 2678-2690, 1999). Further, Bmi1 is known to regulate the expression of target genes by modifying chromatin organization. These functions allow Bmi1 to play an important role in the self-renewal of neural stem cells and hematopoietic stem cells. Based on this, the present inventors succeeded in the reprogramming of astrocytes to induce neural stem cells by overexpressing Bmi1 therein. The induced neural stem cells were similar in many aspects to those isolated from mice. Inter alia, the induced neural stem cells were found to have an increased expression level of Sox2, a gene essential for the self renewal of neural stem cells (Biochem Biophys Res Commun. 371, 267-272, 2008).
Somatic cells require four (Oct4, Sox2, Klf4, C-Myc) or three (Oct4, Sox2, Klf4) genes for their dedifferentiation. It is known that these genes may not be additionally introduced into the cells which endogenously express them. Representatively, it was demonstrated that the introduction of Oct4 alone induces the generation of iPS cells from mouse/human neural cells since they show the endogenous expression of Sox2, Klf4 and C-Myc (Nature, 461, 649-653, 2009). Nowhere has, however, the process of generating pluripotent embryonic stem cell-like cells with Oct4 factor alone been known in the art.